Eukaryotic DNA transposons can be classified into a number of distinct superfamilies, and one of the most widely distributed of these is the so-called hAT superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases (2,3) with Hermes, a hAT transposon that is active not only in the house fly from which it was isolated but also in other insects such as Aedes aegypti, the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae. An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests. Hermes transposition employs a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon, as also seen for the RAG1/2 recombinase of the adaptive immune system. We are continuing our investigation into the mechanism of Hermes DNA binding and hairpin formation using both crystallographic and Cryoelectron microscopy approaches. We have recently determined several co-crystal structures of Hermes bound to DNA that mimics the reaction step just before hairpin formation. This has revealed a large DNA conformational change between the initial cleavage step and subsequent hairpin formation. It seems that two factors affect the conformational change: the divalent metal ions bound at the active site, and the identity of a specific flanking basepair. The structures have also pointed to the catalytic importance of a histidine residue within a conserved C/DxxH motif present in several other transposase families. Another DNA transposition system of interest to us is piggyBac, an active moth transposon. This transposition system arguably has the widest range of current applications by virtue of its ability to function in many cell types and itsseamless insertion/excision mechanism that does not require DNA synthesis to repair its sites of action, unique among transposons. Many aspects of its transposition mechanism are not yet well-understood. We have recently shown that the cysteine-rich carboxy-terminal domain of the piggyBac transposase functions as the site-specific DNA binding domain, and we have collaborated to solve its structure by NMR (4). Guided by the knowledge the structure has provided, we are in the process of biochemically characterizing the precise DNA sequence requirements of piggyBac transposition. The third superfamily of eukaryotic DNA transposons that we have been studying are the Helitrons. Although no currently mobile Helitrons have been identified, they must once have been very active, as their remnants are widespread throughout the eukaryotic kingdom. Unlike other known eukaryotic DNA transposons, Helitron insertions in the host genome are not bordered by target site duplications, suggesting a replicative transposition mechanism that differs substantially from the cut-and-paste mode of transposition used by all other currently characterized eukaryotic systems We have been investigating a reconstituted Helitron transposon, Helraiser (5), and have shown that for transposition, the donor site must be double-stranded and that single-stranded donors do not suffice (6). Nevertheless, replication and integration assays reveal that only one of the transposon donor strands is used. Our investigation of Helitron mobility has established the uniqueness of the Helitron transposition mechanism and suggests its potential in future, novel genomic applications. 2. Hickman et al. (2005) Molecular architecture of a eukaryotic DNA transposase. Nature Struct. Mol. Biol. 12, 715-721. 3. Hickman et al. (2014) Structural basis of transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica. Cell 158, 353-367. 4. Morellet et al. (2018) Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase. Nucleic Acids Res. 46, 2660-2677. 5. Grabundzija et al. (2016) A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes. Nature Commun. 7, 10716. 6. Grabundzija, Hickman, and Dyda (2018) Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition. Nature Commun. 9, 1278.